Breast cancer is a model for the development of effective chemoprevention strategies. The disease has a long latent period that allows biologic modulation to alter its course of development. In addition, the risk factors for breast cancer development are becoming increasingly better-defined allowing the identification of patient populations who would be appropriate for intervention. A population particularly in need of intervention is the pre-menopausal patient whose breast cancers are, in general, estrogen receptor (ER) negative and more aggressive. In fact, despite having breast cancer that may be diagnosed at an early stage, patients with ER negative breast cancer will relapse their disease more frequently than their ER positive counterparts. Vaccines have been one of the most successful clinical interventions for the prevention of human disease. Prospective immunization against a variety of pathogens has resulted in protection from the development of life-threatening infection. In addition, one of the hallmarks of successful immunization is eliciting a specific immune response, therefore, a surrogate marker of vaccine efficacy is well defined. Finally, the clinical endpoint of any vaccine is the generation of immunologic memory; a lifelong endogenous immune response that would prevent the growth of disease at any distant time point after active immunization. For this reason, cancer vaccines have theoretical appeal as a chemopreventive agent in breast cancer. The potential for the development of effective cancer vaccines has increased over the last several years due to the identification of several breast cancer antigens, development of highly quantitative assays for the assessment of the cancer specific immune response after vaccination, and clinically targeting patients with minimal disease burden as candidates for vaccination. The neu transgenic (tg) mouse is a model system for the study and development of multi-antigen vaccines to prevent breast cancer relapse. The mouse develops neu-overexpressing tumors that are ER negative and mediated by neu and cyclin D1 overexpression much like pre-menopausal breast cancers in humans. In addition, neu-tg mice have pre-existent immunity to multiple antigens expressed by their breast cancers. The purpose of using multi-antigen vaccines to immunize against breast cancer rather than strategies targeting a single immunogenic protein is two-fold. First, breast cancer is not caused by a single genetic alteration, but rather a series of initial genetic changes. Secondly, targeting a single tumor antigen will cause immunologic pressure against that protein resulting in the development of antigen negative variants, a phenomenon that has been well described in melanoma and renal cell cancer. Generating immunity against multiple antigens simultaneously may allow the eradication of micrometastatic disease and protection against cancer. The neu-tg mouse provides a pre-clinical system for testing clinical vaccine strategies for breast cancer prevention, evaluation of potential surrogate markers, and identification of breast cancer antigens leading to the development of multi-antigen vaccines targeting overexpressed self-proteins involved in the malignant transformation of breast cancer. The specific aims of this proposal are to: (1) identify and validate appropriate surrogate biomarkers for vaccine efficacy in the chemoprevention of estrogen receptor negative breast cancer, (2) identify potential immunologic targets found in estrogen receptor negative breast cancers for inclusion in a multi-antigen vaccine for breast cancer prevention and, (3) determine the clinical efficacy of multi-antigen vaccines for breast cancer prevention.